Yeast strains and their uses in the production of lipids

ABSTRACT

The invention is directed to isolated microorganisms, as well as biomasses, cultures, microbial oils, and compositions thereof. The invention also provides methods of producing the microbial oils and methods of using the isolated microorganisms, biomasses, and microbial oils.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to isolated microorganisms, as well asbiomasses, cultures, microbial oils, and compositions thereof. Theinvention also provides methods of producing the microbial oils andmethods of using the isolated microorganisms, biomasses, cultures, andmicrobial oils.

2. Background Art

Fatty acids are classified based on the length and saturationcharacteristics of the carbon chain. Fatty acids are termed saturatedfatty acids when no double bonds are present between the carbon atomsand are termed unsaturated fatty acids when double bonds are present.Unsaturated long chain fatty acids are monounsaturated when only onedouble bond is present and are polyunsaturated when more than one doublebond is present.

Polyunsaturated fatty acids (“PUFAs”) are classified based on theposition of the first double bond from the methyl end of the fatty acid:omega-3 (n-3) fatty acids contain a first double bond at the thirdcarbon, while omega-6 (n-6) fatty acids contain a first double bond atthe sixth carbon. For example, docosahexaenoic acid (“DHA”) is anomega-3 long chain polyunsaturated fatty acid (“LC-PUFA”) with a chainlength of 22 carbons and 6 double bonds, often designated as “22:6 n-3.”Other omega-3 LC-PUFAs include eicosapentaenoic acid (“EPA”), designatedas “20:5 n-3,” and omega-3 docosapentaenoic acid (“DPA n-3”), designatedas “22:5 n-3.” DHA and EPA have been termed “essential” fatty acids.Omega-6 LC-PUFAs include arachidonic acid (“ARA”), designated as “20:4n-6,” and omega-6 docosapentaenoic acid (“DPA n-6”), designated as “22:5n-6.”

The production of biological oils from sources such as plants (includingoilseeds), microorganisms, and animals is essential for variouspurposes. For example, it is desirable to increase the dietary intake ofmany beneficial nutrients found in biological oils. Particularlybeneficial nutrients include fatty acids such as omega-3 and omega-6fatty acids and esters thereof. Omega-3 fatty acids are recognized asimportant dietary compounds for preventing arteriosclerosis and coronaryheart disease, for alleviating inflammatory conditions and for retardingthe growth of tumor cells. Omega-6 fatty acids serve not only asstructural lipids in the human body, but also as precursors for a numberof factors in inflammation, such as prostaglandins, leukotrienes, andoxylipins.

Because humans and many other animals cannot directly synthesize omega-3and omega-6 essential fatty acids, they must be obtained in the diet.Traditional dietary sources of essential fatty acids include vegetableoils, marine animal oils, fish oils and oilseeds. In addition, oilsproduced by certain microorganisms have been found to be rich inessential fatty acids.

Oleic acid is another important beneficial fatty acid. Oleic acid is anomega-9 fatty acid that has been associated with health benefits such asslowing the development of heart disease and promoting the production ofantioxidants. It is also used as an ingredient in Lorenzo's oil, amedication developed to prevent the onset of adrenoleukodystrophy (ALD).Oleic acid has also been used as a cosmetic ingredient due to itsmoisturizing effect.

Linoleic acid is also an example of an important fatty acid. It is anunsaturated omega-6 fatty acid that is essential to various biologicalprocesses, such as those involved in hair loss, wound healing, cysticfibrosis, dermatitis, and diabetes. Linoleic acid is also used as acosmetic ingredient due to its beneficial effects on the skin, and inthe making of soaps and emulsifiers.

In order to reduce the costs associated with the production ofbeneficial fatty acids for dietary, pharmaceutical, and cosmetic uses,there exists a need for a low-cost and efficient method of producingbiological oils containing these fatty acids.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an isolated microorganism of thespecies selected from the group consisting of Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorulaingeniosa, wherein the microorganism is capable of producing a microbialoil comprising less than 30% by weight saturated fatty acids.

In some embodiments, the isolated microorganisms are capable ofproducing fatty acids in an amount that is at least 30% by weight of thedry cell weight. In some embodiments, the isolated microorganisms arecapable of producing fatty acids in an amount that is at least 40%, atleast 50%, or at least 60% by weight of the dry cell weight.

In some embodiments, the isolated microorganisms of the invention arecapable of producing a microbial oil comprising greater than 30% byweight, greater than 40% by weight, greater than 50% by weight, orgreater than 60% by weight oleic acid.

In some embodiments, the isolated microorganisms of the invention arecapable of producing a microbial oil comprising greater than 5% byweight, greater than 10% by weight, or greater than 15% by weightlinoleic acid.

In some embodiments, the isolated microorganisms of the invention arecapable of producing a microbial oil comprising less than 25% by weightsaturated fatty acids. In some embodiments, the microorganisms of theinvention are capable of producing a microbial oil comprising less than25% by weight, less than 20% by weight, or less than 15% by weightpalmitic acid.

In some embodiments, the isolated microorganisms of the invention arecapable of producing a microbial oil comprising less than 10% by weight,or less than 5% by weight long chain polyunsaturated fatty acids of 20or more carbon chain length.

The present invention is directed to an isolated microorganism of thespecies selected from the group consisting of Pseudozyma aphidis,Pseudozyma rugulosa, and Rhodotorula ingeniosa, wherein themicroorganism is capable of producing fatty acids in an amount that isat least 30% by weight of the dry cell weight. In some embodiments, theisolated microorganisms of the species selected from the groupconsisting of Pseudozyma aphidis, Pseudozyma rugulosa, and Rhodotorulaingeniosa are capable of producing fatty acids in an amount that is atleast 40% or at least 50% by weight of the dry cell weight.

The present invention is further directed to an isolated microorganismof the Sporidiobolus pararoseus species, wherein the microorganism iscapable of producing fatty acids in an amount that is at least 50% byweight of the dry cell weight. In some embodiments, the isolatedmicroorganisms of the Sporidiobolus pararoseus species are capable ofproducing fatty acids in an amount that is at least 55% or at least 60%by weight of the dry cell weight.

The present invention is directed to an isolated microorganism of thespecies selected from the group consisting of Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorulaingeniosa, wherein the microorganism is capable of producing a microbialoil comprising 0.5% to 30% by weight saturated fatty acids, and whereinthe microorganism is capable of producing fatty acids in an amount thatis 30% to 80% by weight of the dry cell weight.

The present invention is also directed to an isolated microorganism ofthe species selected from the group consisting of Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, and Rhodotorulaingeniosa, wherein the microorganism is capable of producing a microbialoil comprising 30% to 70% by weight oleic acid and 5% to 30% by weightlinoleic acid.

The present invention is further directed to an isolated microorganismof the Sporidiobolus pararoseus species, wherein the microorganism iscapable of producing fatty acids in an amount that is 50% to 80% byweight of the dry cell weight, and wherein the microorganism is capableof producing a microbial oil comprising 5% to 30% by weight linoleicacid.

The present invention is directed to an isolated microorganism depositedunder ATCC Accession No. PTA-11615, an isolated microorganism depositedunder ATCC Accession No. PTA-11616, and an isolated microorganismdeposited under ATCC Accession No. PTA-11617.

The present invention is also directed to an isolated microorganismhaving the characteristics of the microorganism deposited under ATCCAccession No. PTA-11615, an isolated microorganism having thecharacteristics of the microorganism deposited under ATCC Accession No.PTA-11616, and an isolated microorganism having the characteristics ofthe microorganism deposited under ATCC Accession No. PTA-11617.

The present invention is directed to an isolated biomass comprising anisolated microorganism of the invention or mixtures of the isolatedmicroorganisms of the invention.

In some embodiments, at least 30% by weight of the dry cell weight ofthe isolated biomass are fatty acids.

The present invention is also directed to a culture comprising anisolated microorganism of invention or mixtures of the isolatedmicroorganisms of the invention.

In some embodiments, the culture comprises at least 5 g/L of biomass ofthe isolated microorganism.

The present invention is directed to a method for producing a microbialoil, comprising: growing an isolated microorganism of the invention ormixtures of isolated microorganisms of the invention in a culture toproduce a microbial oil.

In some embodiments, the method further comprises extracting themicrobial oil.

In some embodiments, the isolated microorganisms are grown in thepresence of a carbon source selected from the group consisting ofsucrose, glucose, fructose, xylose, glycerol, mannose, arabinose,lactose, galactose, maltose, cellulose, lignocellulose, and combinationsthereof.

In some embodiments, the method produces a culture comprising biomass ofthe isolated microorganism, and the culture comprises at least 5 g/L ofthe biomass.

The present invention is further directed to microbial oils produced bythe methods of the invention, and to the use of the isolatedmicroorganism, biomass, culture, or microbial oil of the invention forthe manufacture of a food, supplement, cosmetic, or pharmaceuticalcomposition for a non-human animal or human.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the DNA sequences of strain 28428 (ATCC Accession No.PTA-11615).

FIG. 2 shows the phylogenetic tree of strain 28428 (ATCC Accession No.PTA-11615) D1/D2 DNA sequences.

FIG. 3 shows the phylogenetic tree of strain 28428 (ATCC Accession No.PTA-11615) ITS DNA sequences.

FIG. 4 shows the DNA sequences of strain 29404 (ATCC Accession No.PTA-11616).

FIG. 5 shows the phylogenetic tree of strain 29404 (ATCC Accession No.PTA-11616) D1/D2 DNA sequences.

FIG. 6 shows the phylogenetic tree of strain 29404 (ATCC Accession No.PTA-11616) ITS DNA sequences.

FIG. 7 shows the DNA sequences of strain 29794 (ATCC Accession No.PTA-11617).

FIG. 8 shows the phylogenetic tree of strain 29794 (ATCC Accession No.PTA-11617) D1/D2 DNA sequences.

FIG. 9 shows the phylogenetic tree of strain 29794 (ATCC Accession No.PTA-11617) ITS DNA sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to isolated microorganism of thePseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, orRhodotorula ingeniosa species, as well as microorganisms having thecharacteristics of the isolated microorganisms, and biomasses, microbialoils, compositions, and cultures thereof. The present invention is alsodirected to methods of producing microbial oils from the isolatedmicroorganisms of the invention, and methods of using the isolatedmicroorganisms, biomasses, cultures, and microbial oils. The isolatedmicroorganisms described herein are highly productive as compared toprior isolates and produce unique fatty acid profiles, characterized inpart by high levels of short-chain unsaturated fatty acids, low levelsof saturated fatty acids, and low levels of long chain polyunsaturatedfatty acids.

In some embodiments, the isolated microorganisms of the invention arecapable of producing fatty acids in an amount that is at least 30% byweight of the dry cell weight. In some embodiments, the isolatedmicroorganisms of the invention are capable of producing fatty acids inan amount that is at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, or at least 65% by weight of the drycell weight. In some embodiments, the isolated microorganisms of theinvention are capable of producing fatty acids in an mount that is 30%to 80% by weight of the dry cell weight, 35% to 80% by weight of the drycell weight, 40% to 80% by weight of the dry cell weight, 45% to 75% byweight of the dry cell weight, 50% to 75% by weight of the dry cellweight, 55% to 70% by weight of the dry cell weight, or 60% to 70% byweight of the dry cell weight.

In some embodiments, the invention is directed to an isolatedmicroorganism of the Pseudozyma aphidis, Pseudozyma rugulosa, orRhodotorula ingeniosa species, wherein the microorganism is capable ofproducing fatty acids in an amount that is at least 30% by weight of thedry cell weight. In some embodiments, an isolated microorganisms of thePseudozyma aphidis, Pseudozyma rugulosa, or Rhodotorula ingeniosaspecies are capable of producing fatty acids in an amount that is atleast 35%, at least 40%, at least 45%, at least 50%, or at least 55% byweight of the dry cell weight. In some embodiments, the isolatedmicroorganisms of the Pseudozyma aphidis, Pseudozyma rugulosa, orRhodotorula ingeniosa species are capable of producing fatty acids in anamount that is 30% to 80% by weight of the dry cell weight, 35% to 75%by weight of the dry cell weight, 40% to 70% by weight of the dry cellweight, 45% to 70% by weight of the dry cell weight, 50% to 65% byweight of the dry cell weight, or 55% to 65% by weight of the dry cellweight.

The present invention is also directed to an isolated microorganism ofthe Sporidiobolus pararoseus species, wherein the microorganism iscapable of producing fatty acids in an amount that is greater than 45%by weight of the dry cell weight. In some embodiments, the isolatedmicroorganisms of the Sporidiobolus pararoseus species are capable ofproducing fatty acids in an amount that is at least 46%, at least 47%,at least 48%, at least 49%, at least 50%, at least 55%, at least 60%, atleast 65%, or at least 70% by weight of the dry cell weight. In someembodiments, the isolated microorganisms of the Sporidiobolus pararoseusspecies are capable of producing fatty acids in an amount that is 30% to85% by weight of the dry cell weight, 40% to 80% by weight of the drycell weight, 45% to 80% by weight of the dry cell weight, 46% to 80% byweight of the dry cell weight, 47% to 80% by weight of the dry cellweight, 48% to 80% by weight of the dry cell weight, 49% to 80% byweight of the dry cell weight, 50% to 80% by weight of the dry cellweight, 55% to 75% by weight of the dry cell weight, 60% to 70% byweight of the dry cell weight, or 65% to 70% by weight of the dry cellweight.

In some embodiments, the invention is directed to an isolatedmicroorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein themicroorganism is capable of producing a microbial oil comprising greaterthan 30% by weight oleic acid (18:1n-9). In some embodiments, theisolated microorganism of the invention is capable of producing amicrobial oil comprising greater than 35%, greater than 40%, greaterthan 45%, greater than 50%, greater than 55%, greater than 60%, orgreater than 65% by weight of oleic acid. In some embodiments, theisolated microorganism of the invention is capable of producing amicrobial oil comprising from 30% to 70% by weight of oleic acid, from35% to 70% by weight of oleic acid, from 40% to 65% by weight of oleicacid, from 45% to 65% by weight of oleic acid, from 50% to 65% by weightof oleic acid, from 55% to 65% by weight of oleic acid, or from 60% to65% by weight of oleic acid.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising greater than 5% byweight linoleic acid (18:2). In some embodiments, the isolatedmicroorganism of the invention is capable of producing a microbial oilcomprising greater than 7%, greater than 10%, greater than 12%, greaterthan 1.5%, greater than 17%, or greater than 20% by weight of linoleicacid. In some embodiments, the isolated microorganism of the inventionis capable of producing a microbial oil comprising from 5% to 30% byweight of linoleic acid, from 7% to 28% by weight of linoleic acid, from10% to 25% by weight of linoleic acid, from 12% to 25% by weight oflinoleic acid, from 15% to 25% by weight of linoleic acid, from 17% to25% by weight of linoleic acid, or from 20% to 23% by weight of linoleicacid.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 30% by weightsaturated fatty acids. In some embodiments, the isolated microorganismof the invention is capable of producing a microbial oil comprising lessthan 25%, less than 20%, less than 15%, less than 10%, less than 8%, orless than 5% by weight of saturated fatty acids. In some embodiments,the isolated microorganism of the invention is capable of producing amicrobial oil comprising from 0.5% to 30% by weight of saturated fattyacids, from 1% to 25% by weight of saturated fatty acids, horn 1% to 20%by weight of saturated fatty acids, from 1% to 15% by weight ofsaturated fatty acids, from 1% to 10% by weight of saturated fattyacids, from 1% to 8% by weight of saturated fatty acids, or from 1% to5% by weight of saturated fatty acids.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 25% by weightpalmitic acid (16:0). In some embodiments, the isolated microorganism ofthe invention is capable of producing a microbial oil comprising lessthan 23%, less than 20%, less than 18%, or less than 15% by weight ofpalmitic acid. In some embodiments, the isolated microorganism of theinvention is capable of producing a microbial oil comprising from 0.5%to 25% by weight of palmitic acid, from 1% to 20% by weight of palmiticacid, from 1% to 18% by weight of palmitic acid, from 1% to 15% byweight of palmitic acid, from 5% to 15% by weight of palmitic acid, orfrom 10% to 15% by weight of palmitic acid.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 10% by weightlong chain polyunsaturated fatty acids (LC-PUFAs) of 20 or more carbonchain length. In some embodiments, the isolated microorganism of theinvention is capable of producing a microbial oil comprising less than8%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, or less than 0.5% by weight of LCPUFAs of 20 or more carbon chainlength. In some embodiments, the isolated microorganism of the inventionis capable of producing microbial oil comprising from 0% to 10% byweight, from 0% to 8% by weight, from 0% to 5% by weight, from 0% to 4%by weight, from 0% to 3% by weight, from 0% to 2% by weight, or from 0%to 1% by weight of LCPUFAs of 20 or more carbon chain length. In someembodiments, the isolated microorganism of the invention is capable ofproducing a microbial oil with no detectable amount of LCPUFAs of 20 ormore carbon chain length.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 10%, less than8%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, or less than 0.5% by weight of docosahexaenoic acid (DHA). In someembodiments, the isolated microorganism of the invention is capable ofproducing a microbial oil comprising from 0% to 10% by weight of DHA,from 0% to 8% by weight of DHA, from 0% to 5% by weight of DHA, from 0%to 4% by weight of DHA, from 0% to 3% by weight of DHA, from 0% to 2% byweight of DHA, or from 0% to 1% by weight of DHA. In some embodiments,the isolated microorganism of the invention is capable of producing amicrobial oil with no detectable amount of DHA.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 1.0%, lessthan 8%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1%, or less than 0.5% by weight of eicosapentaenoic acid (EPA). Insome embodiments, the isolated microorganism of the invention is capableof producing a microbial oil comprising from 0% to 10% by weight of EPA,from 0% to 8% by weight of EPA, from 0% to 5% by weight of EPA, from 0%to 4% by weight of EPA, from 0% to 3% by weight of EPA, from 0% to 2% byweight of EPA, or from 0% to 1% by weight of EPA. In some embodiments,the isolated microorganism of the invention is capable of producing amicrobial oil with no detectable amount of EPA.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 10%, less than8%, than 5%, less than 4%, less than 3%, less than 2%, less than 1%, orless than 0.5% by weight of omega-3 docosapentaenoic acid (DPA n-3). Insome embodiments, the isolated microorganism of the invention is capableof producing a microbial oil comprising from 0% to 10% by weight of DPAn-3, from 0% to 8% by weight of DPA n-3, from 0% to 5% by weight of DPAn-3, from 0% to 4% by weight of DPA n-3, from 0% to 3% by weight of DPAn-3, from 0% to 2% by weight of DPA n-3, or from 0% to 1% by weight ofDPA n-3. In some embodiments, the isolated microorganism of theinvention is capable of producing a microbial oil with no detectableamount of DPA n-3.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 10%, less than8%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, or less than 0.5% by weight of omega-6 docosapentaenoic acid (DPAn-6). In some embodiments, the isolated microorganism of the inventionis capable of producing a microbial oil comprising from 0% to 10% byweight of DPA n-6, from 0% to 8% by weight of DPA n-6, from 0% to 5% byweight of DPA n-6, from 0% to 4% by weight of DPA n-6, from 0% to 3% byweight of DPA n-6, from 0% to 2% by weight of DPA n-6, or from 0% to 1%by weight of DPA n-6. In some embodiments, the isolated microorganism ofthe invention is capable of producing a microbial oil with no detectableamount of DPA n-6.

In some embodiments, the isolated microorganism of the invention iscapable of producing a microbial oil comprising less than 10%, less than8%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, or less than 0.5% by weight of omega-6 arachidonic acid (ARA). Insome embodiments, the isolated microorganism of the invention is capableof producing a microbial oil comprising from 0% to 10% by weight of ARA,from 0% to 8% by weight of ARA, from 0% to 5% by weight of ARA, from 0%to 4% by weight of ARA, from 0% to 3% by weight of ARA, from 0% to 2% byweight of ARA, or from 0% to 1% by weight of ARA. In some embodiments,the isolated microorganism of the invention is capable of producing amicrobial oil with no detectable amount of ARA.

In some embodiments, the invention is directed to an isolatedmicroorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein themicroorganism is capable of producing a microbial oil comprising 0.5% to30% by weight saturated fatty acids, and wherein the microorganism iscapable of producing fatty acids in an amount that is 30% to 80% byweight of the dry cell weight. In some embodiments, the isolatedmicroorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable ofproducing a microbial oil comprising 1% to 25% by weight saturated fattyacids, and is capable of producing fatty acids in an amount that is 40%to 80% by weight of the dry cell weight. In some embodiments, theisolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable ofproducing a microbial oil comprising 1% to 20% by weight saturated fattyacids, and is capable of producing fatty acids in an amount that is 50%to 80% by weight of the dry cell weight. In some embodiments, theisolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable ofproducing a microbial oil comprising 1% to 20% by weight saturated fattyacids, and is capable of producing fatty acids in an amount that is 55%to 75% by weight of the dry cell weight. In some embodiments, theisolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable ofproducing a microbial oil comprising 1% to 20% by weight saturated fattyacids, and is capable of producing fatty acids in an amount that is 60%to 75% by weight of the dry cell weight. In some embodiments, theisolated microorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species is capable ofproducing a microbial oil comprising 1% to 20% by weight saturated fattyacids, and is capable of producing fatty acids in an amount that is 60%to 70% by weight of the dry cell weight.

In some embodiments, the invention is directed to an isolatedmicroorganism of the Pseudozyma aphidis, Pseudozyma rugulosa,Sporidiobolus pararoseus, or Rhodotorula ingeniosa species, wherein themicroorganism is capable of producing a microbial oil comprising 30% to70% by weight oleic acid and 5% to 30% by weight linoleic acid. In someembodiments, the isolated microorganism of the Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosaspecies is capable of producing a microbial oil comprising 35% to 70% byweight oleic acid and 5% to 25% by weight linoleic acid. In someembodiments, the isolated microorganism of the Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosaspecies is capable of producing a microbial oil comprising 40% to 65% byweight oleic acid and 5% to 20% by weight linoleic acid. In someembodiments, the isolated microorganism of the Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosaspecies is capable of producing a microbial oil comprising 45% to 65% byweight oleic acid and 5% to 20% by weight linoleic acid. In someembodiments, the isolated microorganism of the Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosaspecies is capable of producing a microbial oil comprising 50% to 65% byweight oleic acid and 7% to 20% by weight linoleic acid. In someembodiments, the isolated microorganism of the Pseudozyma aphidis,Pseudozyma rugulosa, Sporidiobolus pararoseus, or Rhodotorula ingeniosaspecies is capable of producing a microbial oil comprising 55% to 65% byweight oleic acid and 7% to 15% by weight linoleic acid.

In some embodiments, the invention is directed to an isolatedmicroorganism of the Sporidiobolus pararoseus species, wherein themicroorganism is capable of producing fatty acids in an amount that is50% to 80% by weight of the dry cell weight, and wherein themicroorganism is capable of producing a microbial oil comprising 5% to30% by weight linoleic acid. In some embodiments, the isolatedmicroorganism of the Sporidiobolus pararoseus species is capable ofproducing fatty acids in an amount that is 55% to 75% by weight of thedry cell weight, and wherein the microorganism is capable of producing amicrobial oil comprising 5% to 25% by weight linoleic acid. In someembodiments, the isolated microorganism of the Sporidiobolus pararoseusspecies is capable of producing fatty acids in an amount that is 60% to70% by weight of the dry cell weight, and wherein the microorganism iscapable of producing a microbial oil comprising 7% to 20% by weightlinoleic acid. In some embodiments, the isolated microorganism of theSporidiobolus pararoseus species is capable of producing fatty acids inan amount that is 65% to 70% by weight of the dry cell weight, andwherein the microorganism is capable of producing a microbial oilcomprising 7% to 15% by weight linoleic acid.

In some embodiments, the invention is directed to an isolatedmicroorganism of the species deposited under ATCC Accession No.PTA-11615. The isolated microorganism is also known herein as Pseudozymaaphidis/rugulosa ATCC PTA-11615. The isolated microorganism associatedwith ATCC Accession No. PTA-11615 was deposited under the BudapestTreaty on Jan. 26, 2011 at the American Type Culture Collection, PatentDepository, 10801 University Boulevard, Manassas, Va. 20110-2209.

In some embodiments, the invention is directed to an isolatedmicroorganism of the species deposited under ATCC Accession No.PTA-11616. The isolated microorganism is also known herein asSporidiobolus pararoseus ATCC PTA-11616. The isolated microorganismassociated with ATCC Accession No. PTA-11616 was deposited under theBudapest Treaty on Jan. 26, 2011 at the American Type CultureCollection, Patent Depository, 10801 University Boulevard, Manassas, Va.20110-2209.

In some embodiments, the invention is directed to an isolatedmicroorganism of the species deposited under ATCC Accession No.PTA-11617. The isolated microorganism is also known herein asRhodotorula ingeniosa ATCC PTA-11617. The isolated microorganismassociated with ATCC Accession No. PTA-11617 was deposited under theBudapest Treaty on Jan. 26, 2011 at the American Type CultureCollection, Patent Depository, 10801 University Boulevard, Manassas, Va.20110-2209.

In some embodiments, the invention is directed to an isolatedmicroorganism having the characteristics of the microorganism depositedunder ATCC Accession No. PTA-11615, ATCC Accession No. PTA-11616, orATCC Accession No. PTA-11617. The characteristics of the speciesdeposited under ATCC Accession No. PTA-11615, ATCC Accession No.PTA-11616, or ATCC Accession No. PTA-11617 include its growth andphenotypic properties (examples of phenotypic properties includemorphological and reproductive properties), its physical and chemicalproperties (such as dry weights and lipid profiles), and its genesequences. In some embodiments, the isolated microorganisms of theinvention have substantially identical phenotypic properties of themicroorganism deposited under ATCC Accession No. PTA-11615, ATCCAccession No. PTA-11616, or ATCC Accession No. PTA-11617. In someembodiments, the isolated microorganisms of the invention havesubstantially identical growth properties of the microorganismsdeposited under ATCC Accession No. PTA-11615, ATCC Accession No.PTA-11616, or ATCC Accession No. PTA-11617.

The invention is further directed to an isolated biomass comprising theisolated microorganisms of the invention. An isolated biomass of theinvention is a harvested cellular biomass obtained by any conventionalmethod for the isolation of a microbial biomass.

The biomass of the invention contains a high level of fatty acids. Insome embodiments, at least 30% by weight of the dry cell weight of thebiomass of the invention are fatty acids. In some embodiments, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least65% by weight of the dry cell weight of the biomass of the invention arefatty acids. In some embodiments, the biomass of the invention comprisesfrom 30% to 75%, from 40% to 75%, from 45% to 70%, from 50% to 70%, from55% to 65% fatty acids by the dry cell weight of the biomass.

The invention is further directed to a culture comprising one or moreisolated microorganisms of the invention. Various fermentationparameters for inoculating, growing, and recovering yeast strains areknown in the art. Any conventional medium for growth of yeasts can beused.

The cultures of the invention contain a high amount of biomass per literof culture broth, indicating efficient growth of the isolatedmicroorganisms. In some embodiments, the cultures comprise at least 1g/L of biomass of the isolated microorganisms of the invention. In someembodiments, the cultures comprise at least 2 g/L, at least 3 g/L, atleast 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8g/L, at least 9 g/L, or at least 10 g/L of biomass of the isolatedmicroorganisms of the invention. In some embodiments, the culturescomprises 1 g/L to 10 g/L, 2 g/L to 10 g/L, 3 g/L to 10 g/L, 4 g/L to 10g/L, 5 g/L to 10 g/L, 6 g/L to 10 g/L, 7 g/L to 10 g/L, 8 g/L to 10 g/L,or 9 g/L to 10 g/L of biomass of the isolated microorganisms of theinvention.

The present invention is further directed to methods of producingmicrobial oils.

In some embodiments, the method comprises growing an isolatedmicroorganism of the invention or mixtures thereof in a culture toproduce a microbial oil. The isolated microorganisms of the inventioncan be grown in the presence of various carbon sources, including, forexample, sucrose, glucose, fructose, xylose, glycerol, mannose,arabinose, lactose, galactose, maltose, cellulose, lignocellulose, orcombinations thereof. The method can further comprise extracting themicrobial oil. The oil can be extracted from a freshly harvested biomassor can be extracted from a previously harvested biomass that has beenstored under conditions that prevent spoilage, Known methods can be usedto culture a microorganism of the invention, to isolate a biomass fromthe culture, to extract a microbial oil from the biomass, and to analyzethe fatty acid profile of oils extracted from the biomass.

The invention is further directed to a microbial oil produced by themethods of the invention. In some embodiments, the microbial oilcomprises a fatty acid profile having the same characteristics as thefatty acid profile of the isolated microorganisms of the invention. Amicrobial oil of the invention can be any oil derived from amicroorganism, including, for example: a crude oil extracted from thebiomass of the microorganism without further processing; a refined oilthat is obtained by treating a crude microbial oil with furtherprocessing steps such as refining, bleaching, and/or deodorizing; adiluted microbial oil obtained by diluting a crude or refined microbialoil; or an enriched oil that is obtained, for example, by treating acrude or refined microbial oil with further methods of purification toincrease the concentration of a fatty acid in the oil.

The invention is also directed to the use of the isolated microorganism,biomass, culture, or microbial oil of the invention far the manufactureof a food, dietary supplement, cosmetic, or pharmaceutical compositionfor a non-human animal or human.

Compositions

The invention is further directed to compositions comprising an isolatedmicroorganism of the invention, an isolated biomass of the invention, amicrobial oil of the invention, or combinations thereof.

An isolated microorganism, biomass, or microbial oil of the inventioncan be further chemically or physically modified or processed based onthe requirements of the composition by any known technique.

Microorganism cells or biomasses can be dried prior to use in acomposition by methods including, but not limited to, freeze drying, airdrying, spray drying, tunnel drying, vacuum drying (lyophilization), ora similar process. Alternatively, a harvested and washed biomass can beused directly in a composition without drying.

Microbial oils of the invention can be used as starting material to moreefficiently produce a product enriched in a fatty acid. For example, themicrobial oils of the invention can be subjected to various purificationtechniques known in the art, such as distillation or urea adduction, toproduce a higher potency product with higher concentrations of aparticular fatty acid. The microbial oils of the invention can also beused in chemical reactions to produce compounds derived from fatty acidsin the oils, such as esters and salts of a fatty acid.

A composition of the invention can include one or more excipients. Asused herein, “excipient” refers to a component, or mixture ofcomponents, that is used in a composition of the present invention togive desirable characteristics to the composition, including foods aswell as pharmaceutical, cosmetic, and industrial compositions. Anexcipient of the present invention can be described as a“pharmaceutically acceptable” excipient when added to a pharmaceuticalcomposition, meaning that the excipient is a compound, material,composition, salt, and/or dosage form which is, within the scope ofsound medical judgment, suitable for contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problematic complications over the desired durationof contact commensurate with a reasonable benefit/risk ratio. In someembodiments, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized internationalpharmacopeia for use in animals, and more particularly in humans.Various excipients can be used. In some embodiments, the excipient canbe, but is not limited to, an alkaline agent, a stabilizer, anantioxidant, an adhesion agent, a separating agent, a coating agent, anexterior phase component, a controlled-release component, a solvent, asurfactant, a humectant, a buffering agent, a filler, an emollient, orcombinations thereof. Excipients in addition to those discussed hereincan include excipients listed in, though not limited to, Remington: TheScience and Practice of Pharmacy, 21^(st) ed. (2005). Inclusion of anexcipient in a particular classification herein (e.g., “solvent”) isintended to illustrate rather than limit the role of the excipient. Aparticular excipient can fall within multiple classifications.

Compositions of the invention include, but are not limited to, foodproducts, pharmaceutical compositions, cosmetics, and industrialcompositions.

In some embodiments, the composition is a food product. A food productis any food for animal or human consumption, and includes both solid andliquid compositions. A food product can be an additive to animal orhuman foods. Foods include, but are not limited to common foods; liquidproducts, including milks, beverages, therapeutic drinks, andnutritional drinks; functional foods; supplements; nutraceuticals;infant formulas, including formulas for pre-mature infants; foods forpregnant or nursing women; foods for adults; geriatric foods; and animalfoods.

In some embodiments, an isolated microorganism, biomass, or microbialoil of the invention can be used directly as or included as an additivewithin one or more of: an oil, shortening, spread, other fattyingredient, beverage; sauce, dairy-based or soy-based food (such asmilk, yogurt, cheese and ice-cream), a baked good, a nutritionalproduct, e.g., as a nutritional supplement (in capsule or tablet form),a vitamin supplement, a diet supplement, a powdered drink, a finished orsemi-finished powdered food product, and combinations thereof.

A partial list of food compositions that can include a microbial oil ofthe invention includes, but is not limited to, soya based products(milks, ice creams, yogurts, drinks, creams, spreads, whiteners); soupsand soup mixes; doughs, batters, and baked food items including, forexample, fine bakery wares, breakfast cereals, cakes, cheesecakes, pies,cupcakes, cookies, bars, breads, rolls, biscuits, muffins, pastries,scones, croutons, crackers, sweet goods, snack cakes, pies,granola/snack bars, and toaster pastries; candy; hard confectionery;chocolate and other confectionery; chewing gum; liquid food products,for example milks, energy drinks, infant formula, carbonated drinks,teas, liquid meals, fruit juices, fruit-based drinks, vegetable-baseddrinks; multivitamin syrups, meal replacers, medicinal foods, andsyrups; powdered beverage mixes; pasta; processed fish products;processed meat products; processed poultry products; gravies and sauces;condiments (ketchup, mayonnaise, etc.); vegetable oil-based spreads;dairy products; yogurt; butters; frozen dairy products; ice creams;frozen desserts; frozen yogurts; semi-solid food products such as babyfood; puddings and gelatin desserts; processed and unprocessed cheese;pancake mixes; food bars including energy bars; waffle mixes; saladdressings; replacement egg mixes; nut and nut-based spreads; saltedsnacks such as potato chips and other chips or crisps, corn chips,tortilla chips, extruded snacks, popcorn, pretzels, potato crisps, andnuts; specialty snacks such as dips, dried fruit snacks, meat snacks,pork rinds, health food bars and rice/corn cakes.

In some embodiments, a microbial oil of the invention can be used tosupplement infant formula. Infant formula can be supplemented with amicrobial oil of the invention alone or in combination with a physicallyrefined oil derived from an arachidonic acid (ARA)-producingmicroorganism.

In some embodiments, the composition is an animal feed. An “animal”means any non-human organism belonging to the kingdom Animalia, andincludes, without limitation, aquatic animals and terrestrial animals.The term “animal feed” or “animal food” refers to any food intended fornon-human animals, whether for fish; commercial fish; ornamental fish;fish larvae; bivalves; mollusks; crustaceans; shellfish; shrimp; larvalshrimp; artemia; rotifers; brine shrimp; filter feeders; amphibians;reptiles; mammals; domestic animals; farm animals; zoo animals; sportanimals; breeding stock; racing animals; show animals; heirloom animals;rare or endangered animals; companion animals; pet animals such as dogs,cats, guinea pigs, rabbits, rats, mice, or horses; primates such asmonkeys (e.g., cebus, rhesus, African green, patas, cynomolgus, andcercopithecus), apes, orangutans, baboons, gibbons, and chimpanzees;canids such as dogs and wolves; felids such as cats, lions, and tigers;equids such as horses, donkeys, and zebras; food animals such as cows,cattle, pigs, and sheep; ungulates such as deer and giraffes; rodentssuch as mice, rats, hamsters and guinea pigs; and so on. An animal feedincludes, but is not limited to, an aquaculture feed, a domestic animalfeed including pet feed, a zoological animal feed, a work animal feed, alivestock feed, or a combination thereof.

In some embodiments, the composition is a feed or feed supplement forany animal whose meat or products are consumed by humans, such as anyanimal from which meat, eggs, or milk is derived for human consumption.When fed to such animals, nutrients such as certain fatty acids can beincorporated into the flesh, milk, eggs or other products of suchanimals to increase their content of these nutrients.

In some embodiments, the composition is a spray-dried material that canbe crumbled to form particles of an appropriate size for consumption byzooplankton, artemia, rotifers, and filter feeders. In some embodiments,the zooplankton, artemia, or rotifers fed by the composition are in turnfed to fish larvae, fish, shellfish, bivalves, or crustaceans.

In some embodiments, the composition is a pharmaceutical composition.Suitable pharmaceutical compositions include, but are not limited to, ananti-inflammatory composition, a drug for treatment of coronary heartdisease, a drug for treatment of arteriosclerosis, a chemotherapeuticagent, an active excipient, an osteoporosis drug, an anti-depressant, ananti-convulsant, an anti Helicobacter pylori drug, a drug for treatmentof neurodegenerative disease, a drug for treatment of degenerative liverdisease, an antibiotic, a cholesterol lowering composition, and atriglyceride lowering composition. In some embodiments, the compositionis a medical food. A medical food includes a food that is in acomposition to be consumed or administered externally under thesupervision of a physician and that is intended for the specific dietarymanagement of a condition, for which distinctive nutritionalrequirements, based on recognized scientific principles, are establishedby medical evaluation.

in some embodiments, the microbial oil can be formulated in a dosageform. Dosage forms can include, but are not limited to, tablets,capsules, cachets, pellets, pills, powders and granules, and parenteraldosage forms, which include, but are not limited to, solutions,suspensions, emulsions, and dry powders comprising an effective amountof the microbial oil. It is also known in the art that such formulationscan also contain pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. Administration forms caninclude, but are not limited to, tablets, dragees, capsules, caplets,and pills, which contain the microbial oil and one or more suitablepharmaceutically acceptable carriers.

For oral administration, the microbial oil can be combined withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the microbial oils of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.In some embodiments, the dosage form is a tablet, pill or caplet.Pharmaceutical preparations for oral use can be obtained by adding asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude, but are not limited to, fillers such as sugars, including, butnot limited to, lactose, sucrose, mannitol, and sorbitol; cellulosepreparations such as, but not limited to, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, andpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate, Pharmaceutical preparations that can be used orally include,but are not limited to, push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a plasticizer, such asglycerol or sorbitol.

In some embodiments, the composition is a cosmetic. Cosmetics include,but are not limited to, emulsions, creams, lotions, masks, soaps,shampoos, washes, facial creams, conditioners, make-ups, bath agents,and dispersion liquids. Cosmetic agents can be medicinal ornon-medicinal.

In some embodiments, the composition is an industrial composition. Insome embodiments, the composition is a starting material for one or moremanufactures. A manufacture includes, but is not limited to, a polymer;a photographic photosensitive material; a detergent; an industrial oil;or an industrial detergent. For example, U.S. Pat. No. 7,259,006describes use of DHA-containing fat and oil for production of behenicacid and production of photographic sensitive materials using behenicacid.

In some embodiments, the composition is a lipid-based biofuelmanufactured by converting the biological oil of the invention intolipid-based biofuel through known means in the art, such as throughtransesterifying the biological oil to produce biodiesel, Different usesof the biological oils of the present invention for lipid-based biofuelpurposes include, but are not limited to, uses as heating oil,transportation biodiesel, jet fuel, fuel additives, specialty fuels andlubricants. In some embodiments, the conversion of biological oils intolipid-based biofuels involves chemical processes and refining techniquesknown in the art which may also produce or be used to produce specialtychemical compounds similar to petroleum, distillates.

Kits Comprising the Compositions

The invention is further directed to kits or packages containing one ormore units of a composition of the invention. Kits or packages caninclude units of a food product, pharmaceutical composition, cosmetic,or industrial composition comprising the isolated microorganism,biomass, or microbial oil of the invention, or combinations thereof.Kits or packages can also include an additive comprising the isolatedmicroorganism, biomass, or microbial oil of the invention, orcombinations thereof for preparation of a food, cosmetic, pharmaceuticalcomposition, or industrial composition.

In some embodiments, the kit or package contains one or more units of apharmaceutical composition to be administered according to the methodsof the present invention. The kit or package can contain one dosageunit, or more than one dosage unit (i.e., multiple dosage units). Ifmultiple dosage units are present in the kit or package, the multipledosage units can be optionally arranged for sequential administration.

The kits of the present invention can optionally contain instructionsassociated with the units or dosage forms of the kits. Such instructionscan be in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceutical products, which noticereflects approval by the agency of the manufacture, use or sale forhuman administration to treat a condition or disorder. The instructionscan be in any form which conveys information on the use of the units ordosage forms in the kit according to the methods of the invention. Forexample, the instructions can be in the form of printed matter, or inthe form of a pre-recorded media device.

In the course of examination of a patient, a medical professional candetermine that administration of one of the methods of the presentinvention is appropriate for the patient, or the physician can determinethat the patient's condition can be improved by the administration ofone of the methods of the present invention. Prior to prescribing anyregimen, the physician can counsel the patient, for example, on thevarious risks and benefits associated with the regimen. The patient canbe provided full disclosure of all known and suspected risks associatedwith the regimen. Such counseling can be provided verbally, as well asin written form. In some embodiments, the physician can provide thepatient with literature materials on the regimen, such as productinformation, educational materials, and the like.

The present invention is also directed to methods of educating consumersabout the methods of treatment, the method comprising distributing thedosage forms with consumer information at a point of sale. In someembodiments, the distribution will occur at a point of sale having apharmacist or healthcare provider.

The term “consumer information” can include, but is not limited to, anEnglish language text, non-English language text, visual image, chart,telephone recording, website, and access to a live customer servicerepresentative. In some embodiments, consumer information will providedirections for use of the dosage forms according to the methods of thepresent invention, appropriate age use, indication, contraindications,appropriate dosing, warnings, telephone number of website address. Insome embodiments, the method further comprises providing professionalinformation to relevant persons in a position to answer consumerquestions regarding use of the disclosed regimens according to themethods of the present invention. The term “professional information”includes, but is not limited to, information concerning the regimen whenadministered according to the methods of the present invention that isdesigned to enable a medical professional to answer customer questions.

A “medical professional,” includes, for example, a physician, physicianassistant, nurse practitioner, pharmacist and customer servicerepresentative.

EXAMPLES Example 1

Identifications were determined for three yeast strains: ATCC AccessionNo. PTA-11615 (Strain 28428), ATCC Accession No. PTA-11616 (Strain29404) and ATCC Accession. No, PTA-11617 (Strain 29794). The yeasts wereplated on Malt Agar, DNA extractions were conducted and ribosomal genesequence analysis performed. Sequence homology comparisons were donebetween the D1D2 and ITS regions and known DNA sequences from yeaststrains in the public databases. Morphological examination was alsoconducted for comparisons to known yeast species.

Strain 28428 DNA sequences (FIG. 1) matched 100% with the Genbanksequence for the Type strain of Pseudozyma aphidis in the first regionanalyzed (D1/D2 domains of the Large subunit) and differed by 1 mismatchfrom the Type strain of Pseudozyma rugulosa (FIG. 2). In the secondregion (ITS) out of 545 possible matches, the strain showed 2 mismatchesfrom the Type strain of P. aphidis and again 1 from the Type strain ofP. rugulosa (FIG. 3). The two species appear to be closely relatedshowing genetic variability within and between them. Morphologicalcharacteristics are also very similar between P. aphidis and P. rugulosaand matched the morphology observations in strain 28428, which showedflat, dull, yellowish-cream colonies with fringed margin and fusiformcells, variable in size, with polar budding on short denticles. Based onthe data in hand, it is not possible to distinguish the identificationmore specifically than as either P. aphidis or P. rugulosa.

Strain 29404 DNA sequences (FIG. 4) matched perfectly in both regionswith the Genbank sequences for Sporidiobolus pararoseus CBS 484,Comparison to the Type strain of the species CBS 491 had 2 mismatches inD1D2 and 2 in ITS (FIGS. 5 & 6). Morphological characteristics (ovoidcells, single or in short chains, and shiny coral red colonies with asmooth surface and an entire margin) confirmed the identification. Theidentification of this strain is Sporidiobolus pararoseus.

Strain 29794 DNA sequences (FIG. 7) showed an identical sequence matchto the type strain of Rhodotorula ingeniosa (CBS 4240) in the D1D2domains (FIG. 8) and only one mismatch out of 590 possible matches inthe ITS regions (FIG. 9), which is possibly due to intra-specificvariability. Cell and colony morphology (ovoid to cylindrical cells,single or in pairs, a thin capsule and yellowish, glistening, highlymucoid colonies) confirmed the identification. The identification ofthis strain is R. ingeniosa.

Example 2

Isolated microorganisms of Sporidiobolus pararoseus and Rhodotorulaingeniosa were grown in ¼X BFGM medium (Table 1). Each strain was pickedfrom an agar plate, and inoculated into a shake flask. The shake flaskwas then used to inoculate another flask (250 ml Erlenmeyer flaskcontaining 50 ml of medium) that was then grown for 7 days. After 7 daysthe flask was harvested by centrifugation, the pellet was washed withwater and centrifuged again. The final pellet was freeze dried, theweight was measured and then total fat (total fatty acids) and fattyacid profile were determined by FAME procedure.

The isolated microorganism of Pseudozyma aphidis/rugulosa was grown inthe media below (Media 2) and the fatty acid profile was determined.Results of the fatty acid profile analysis fir all three strains areshown in Table 2.

Media 2:

15 g/L Reef Crystal Artificial Sea salts

1 g/L glucose

1 g/L monosodium glutamate

0.2 g/L yeast extract

1 mL/L vitamin mix*

mL/L PII trace metal mix**

0.1 g/L penicillin G

0.1 g/L streptomycin sulfate

*vitamin mix contains 100 mg/L thiamine, 0.5 mg/L biotin, 0.5 g/Lcyanocobalmin**PII trace metal mix contains:

-   -   6.0 g/L Na₂EDTA    -   0.29 g/L FeCl₃.6H₂O    -   6.84 g/L H₃BO₃    -   0.86 g/L MnCl₂.4H₂O    -   0.06 g/L ZnCl₂    -   0.026 g/L CoCl₂.6H₂O    -   0.052 g/L NiSO₄.6H₂O    -   0.002 g/L CuSO₄.5H₂O    -   0.005 g/L Na₂MoO₄.2H₂O

TABLE 1 ¼ × BFGM Medium mL of Amount stock to per liter [Stock] use perg/l mg/l Component (g) (g/l) liter Na K Mg Ca Cl Fe Cu Mn Co Zn NaCl0.625 dry 0.25 0.38 KCl 1 50 20 ml 0.52 0.48 MgSO4•7H2O 5 227 22 ml 0.47(NH4)2SO4 0.05 190 0.2625 ml CaCl2 2H2O 0.29 dry 0.08 0.14 MSG 0.5 drymonohydrate Tastone 154 0.5 dry HEPES 23.8 dry (100 mM) pH 7 KH2PO40.025 56.5 0.4425 ml add after autoclaving Sucrose 50 500 100 ml addafter autoclaving Trace Metals see 2 ml add after autoclaving belowVitamins see 1 ml add after autoclaving below Trace Metal SolutionCitric Acid 1.0 g dry FeSO4•7H2O 10.3 mg 5.15 2.07 MnCl2•4H2O 3.1 mg1.55 0.86 ZnSO4•7H2O 1.93 mg 0.965 0.44 CoCl2•6H2O 0.04 mg 0.02 0.01Na2MoO4•2H2O 0.04 mg 0.02 CuSO4•5H2O 2.07 mg 1.035 0.53 NiSO4•6H2O 2.07mg 1.035 pH to 2.5 with HCl Vitamin Solution Vitamin B12 0.16 mg 0.16Thiamine 9.75 mg 9.75 CaPantothenate 3.33 mg 3.33 Ion Totals (ppm)995.8536

TABLE 2 Media ¼ BFGM Media 2 Microorganism Rhodotorula SporidiobolusPseudozyma ingeniosa pararoseus aphidis/rugulosa Sugar Sucrose XyloseSucrose Glucose Temperature 27 27 22.5 30° C. (° C.) Biomass g/L 9.5065.338 8.79 pH 6.9 6.78 6.68 % 08:0 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00% 10:0 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 % 12:00.00 0.00 0.00 % 12:1 0.00 0.00 0.00 % 13:0 0.17 0.18 0.00 % 13:1 0.000.00 0.00 % 14:0 0.58 0.73 0.00 0.8 % 14:1 0.00 0.00 0.00 Trace (<1.0%)% 15:1 0.00 0.00 0.00 % 16:0 18.14 19.62 16.02 21.9 % 16:1 0.64 0.690.29 6.6 % 16:2 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 % 17:0 0.00 0.000.11 % 18:0 3.88 4.13 4.86 2.1 % 18:1 n-9 61.79 59.54 60.78 40.4 % 18:1n-7 0.43 0.19 0.00 % 18:2 8.83 9.76 9.75 24.7 % 18:3 n-6 0.06 0.00 0.00% 18:3 n-3 2.71 2.54 4.72 % 18:4 n-3 0.00 0.00 0.00 % 20:0 1.18 0.920.00 0.8 % 20:1 n-9 0.29 0.30 0.00 0.5 % 20:2 0.00 0.00 0.00 % 20:3 n-90.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 % 20:3 n-3 0.00 0.00 0.00 %20:4 ARA 0.00 0.14 0.00 % 20:5 n-3 0.00 0.00 0.00 EPA % 22:0 0.54 0.550.00 1.1 % 22:1 0.00 0.00 0.00 % 22:2 0.17 0.21 0.00 % 22:3 0.00 0.000.00 % 22:4 n-6 0.00 0.00 0.00 % 22:5 n-6 0.00 0.00 0.00 % 22:5 n-3 0.000.00 0.00 % 22:6 n-3 0.00 0.00 0.00 DHA % 24:0 0.21 0.20 0.00 1.1 % 24:10.00 0.00 0.00 % Fat 55.19 45.79 65.17 % Unknown 0.38 0.31 3.46 08:00.00 0.00 0 09:0 0.00 0.00 0 10:0 0.00 0.00 0 11:0 0.00 0.00 0 11:1 0.000.00 12:0 0.00 0.00 0 12:1 0 13:0 6.04 5.70 0 13:1 0.00 0.00 0 14:020.92 22.93 0 14:1 0.00 0.00 0 15:0 INT 681.22 684.35 112753 STD 15:10.00 0.00 0 16:0 654.11 617.77 110650 16:1 23.02 21.84 2025 16:2 16:317:0 0.00 0.00 759 18:0 140.00 129.99 33583 18:1 n-9 2227.57 1874.95419870 18:1 n-7 15.60 6.08 0 18:2 318.37 307.24 67375 18:3 n-6 2.23 0.000 18:3 n-3 97.78 79.91 32596 18:4 n-3 20:0 42.43 28.86 0 20:1 n-9 10.389.59 0 20:2 0.00 0.00 0 20:3 n-9 20:3 n-6 0.00 0.00 0 20:3 n-3 0.00 0.000 20:4 ARA 0.00 4.40 0 20:5 n-3 0.00 0.00 0 EPA 22:0 19.48 17.18 0 22:10.00 0.00 0 22:2 6.08 6.67 0 22:3 0.00 0.00 0 22:4 n-6 0.00 0.00 0 22:5n-6 0.00 0.00 0 22:5 n-3 0.00 0.00 0 22:6 n-3 0.00 0.00 0 DHA 23:0 INT0.00 0.00 0 STD 24:0 7.55 6.45 0 24:1 0.00 0.00 0 Total Area 4286.433833.56 803514 Total 3605.22 3149.22 690761

Example 3

Isolated microorganisms having matching DNA sequences to that of ATCCAccession No, PTA-11615 (Pseudozyma aphidis/rugulosa; Strain 28428),ATCC Accession No. PTA-11616 (Sporidiobolus pararoseus; Strain 29404)and ATCC Accession No. PTA-11617 (Rhodotorula ingeniosa; Strain 29794)were grown and the total fat (total fatty acids) and fatty acid profileswere determined. Results are shown in Tables 3 and 4.

TABLE 3 ID Rhodotorula ingeniosa Sporidiobolus pararoseus Strain # 2976429764 29664 29405 29412 29459 29556 29568 29573 Dry Weight (g/l) 4.2311.054 6.44 9.89 6.23 6.86 4.85 1.3 8.46 Medium ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ BFGMBFGM BFGM BFGM BFGM BFGM BFGM BFGM BFGM Sugar Xylose Sucrose sucrosesucrose sucrose sucrose sucrose sucrose sucrose  8:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00  9:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 11:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 12:0 0.00 0.07 0.00 0.00 0.00 0.00 0.03 0.00 0.00 12:10.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 13:0 0.22 0.15 0.09 0.120.13 0.09 0.13 0.18 0.15 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 14:0 0.81 0.71 0.00 0.61 0.85 0.65 0.86 0.80 0.88 14:1 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 15:1 0.00 0.00 0.03 0.00 0.00 0.000.00 0.11 0.00 16:0 21.71 22.03 16.96 16.52 18.79 16.01 19.53 23.8219.22 16:1 0.57 0.66 0.44 0.32 0.20 0.21 0.34 0.50 0.41 16:2 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 16:3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 17:0 0.00 0.00 0.17 0.19 0.09 0.17 0.13 0.18 0.14 18:06.58 6.27 4.04 4.67 7.70 5.83 5.72 10.66 4.30 18:1n9 52.63 53.65 60.3159.97 0.00 62.94 56.56 40.16 59.19 18:1n7 0.28 0.59 0.00 0.00 61.36 0.000.00 0.00 0.00 18:2 10.99 10.52 11.53 9.96 5.84 8.84 11.03 16.40 9.8918:3n6 0.08 0.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18:3n3 3.73 2.762.15 5.24 1.97 1.59 2.78 4.19 2.86 18:4n3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 20:0 0.84 1.14 0.79 0.68 0.83 0.78 0.87 0.38 0.68 20:1n90.26 0.23 0.31 0.06 0.29 0.35 0.22 0.08 0.09 20:2 0.00 0.00 0.05 0.000.00 0.00 0.00 0.00 0.00 20:3n9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 20:3n6 0.00 0.00 0.12 0.11 0.00 0.13 0.10 0.14 0.11 20:3n3 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:4n6 ARA 0.20 0.11 0.00 0.000.00 0.00 0.00 0.00 0.00 20:5n3 EPA 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 22:0 0.64 0.50 1.14 0.64 0.94 1.27 0.88 0.69 0.91 22:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22:2 0.18 0.13 0.00 0.00 0.000.00 0.00 0.00 0.00 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0022:4n6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22:5n6 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 22:5n3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 22:6n3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 24:00.29 0.19 0.00 0.43 0.49 0.84 0.41 1.72 0.58 24:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 Fat 39.04 52.86 67.01 56.02 59.60 63.32 64.4440.38 64.00 Unknown 0.00 0.23 1.88 0.48 0.51 0.30 0.43 0.00 0.58 IDSporidiobolus pararoseus Strain # 29577 29588 29591 29594 29631 2965529596 29631 29655 Dry Weight (g/l) 3.87 5.46 6.31 7.3 4.73 9.41 8.124.73 9.41 Medium ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ BFGM BFGM BFGM BFGM BFGM BFGM BFGMBFGM BFGM Sugar sucrose sucrose sucrose sucrose sucrose sucrose sucrosesucrose sucrose  8:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  9:00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 12:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 13:0 0.27 0.18 0.16 0.14 0.21 0.15 0.38 0.21 0.15 13:10.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 14:0 0.55 0.63 0.57 0.940.53 0.86 0.57 0.53 0.86 14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 15:1 0.14 0.07 0.00 0.04 0.12 0.00 0.11 0.12 0.00 16:0 20.04 18.5016.84 19.22 18.33 19.98 17.98 18.33 19.98 16:1 0.33 0.41 0.34 0.60 0.480.41 0.31 0.48 0.41 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0016:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 17:0 0.33 0.21 0.220.16 0.20 0.16 0.26 0.20 0.16 18:0 5.14 3.67 4.42 3.34 2.37 5.06 5.482.37 5.06 18:1n9 42.35 57.13 57.60 60.19 56.53 54.71 46.89 56.53 54.7118:1n7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18:2 25.32 14.1714.55 10.72 15.84 13.05 21.79 15.84 13.05 18:3n6 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 18:3n3 2.67 1.59 2.87 1.55 2.03 3.52 3.02 2.033.52 18:4n3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:0 0.42 0.550.36 0.57 0.56 0.72 0.72 0.56 0.72 20:1n9 0.16 0.09 0.09 0.08 0.27 0.180.18 0.27 0.18 20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:3n90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:3n6 0.21 0.17 0.13 0.120.20 0.00 0.00 0.20 0.00 20:3n3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 20:4n6 ARA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:5n3 EPA0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22:0 0.63 0.96 0.52 0.880.90 0.73 0.78 0.90 0.73 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22:3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 22:4n6 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 22:5n6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0022:5n3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22:6n3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 24:0 1.02 0.76 0.52 0.76 0.68 0.340.85 0.68 0.34 24:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Fat29.39 43.32 52.30 62.44 33.57 53.57 26.03 33.57 53.57 Unknown 0.41 0.920.79 0.69 0.75 0.14 0.67 0.75 0.14

TABLE 4 Pseudozyma aphidis/rugulosa Strain #: Strain 28426 Media: Media2 Sugar: Glucose % total fatty acids Fatty acid (area %) 14:0 14:1 16:027.4 16:1 1.4 18:0 9.6 18.1 31.5 18:2 28.0 20:0 0.4 20:1 1.6 22:0 0.224:0

All of the various aspects, embodiments, and options described hereincan be combined in any and all variations.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1. An isolated microorganism of the species selected from the groupconsisting of Pseudozyma aphidis, Pseudozyma rugulosa, Sporidioboluspararoseus, and Rhodotorula ingeniosa, wherein the microorganism iscapable of producing a microbial oil comprising less than 30% by weightsaturated fatty acids.
 2. The isolated microorganism of claim 1, whereinthe microorganism is capable of producing fatty acids in an amount thatis at least 30% by weight of the dry cell weight.
 3. The isolatedmicroorganism of claim 1, wherein the microorganism is capable ofproducing fatty acids in an amount that is at least 40% by weight of thedry cell weight.
 4. The isolated microorganism of claim 1, wherein themicroorganism is capable of producing fatty acids in an amount that isat least 50% by weight of the dry cell weight.
 5. The isolatedmicroorganism of claim 1, wherein the microorganism is capable ofproducing fatty acids in an amount that is at least 60% by weight of thedry cell weight.
 6. The isolated microorganism of claim 1, wherein themicroorganism is capable of producing a microbial oil comprising greaterthan 30% by weight oleic acid.
 7. The isolated microorganism of claim 1,wherein the microorganism is capable of producing a microbial oilcomprising greater than 40% by weight oleic acid.
 8. The isolatedmicroorganism of claim 1, wherein the microorganism is capable ofproducing a microbial oil comprising greater than 50% by weight oleicacid.
 9. The isolated microorganism of claim 1, wherein themicroorganism is capable of producing a microbial oil comprising greaterthan 60% by weight oleic acid.
 10. The isolated microorganism of claim1, wherein the microorganism is capable of producing a microbial oilcomprising greater than 5% by weight linoleic acid.
 11. The isolatedmicroorganism of claim 1, wherein the microorganism is capable ofproducing a microbial oil comprising greater than 10% by weight linoleicacid.
 12. The isolated microorganism of claim 1, wherein themicroorganism is capable of producing a microbial oil comprising greaterthan 15% by weight linoleic acid.
 13. The isolated microorganism ofclaim 1, wherein the microorganism is capable of producing a microbialoil comprising less than 25% by weight saturated fatty acids.
 14. Theisolated microorganism of claim 1, wherein the microorganism is capableof producing a microbial oil comprising less than 25% by weight palmiticacid.
 15. The isolated microorganism of claim 1, wherein themicroorganism is capable of producing a microbial oil comprising lessthan 20% by weight palmitic acid.
 16. The isolated microorganism ofclaim 1, wherein the microorganism is capable of producing a microbialoil comprising less than 15% by weight palmitic acid.
 17. The isolatedmicroorganism of claim 1, wherein the microorganism is capable ofproducing a microbial oil comprising less than 10% by weight long chainpolyunsaturated fatty acids of 20 or more carbon chain length.
 18. Theisolated microorganism of claim 1, wherein the microorganism is capableof producing a microbial oil comprising less than 5% by weight longchain polyunsaturated fatty acids of 20 or more carbon chain length. 19.An isolated microorganism of the species selected from the groupconsisting of Pseudozyma aphidis, Pseudozyma rugulosa, and Rhodotorulaingeniosa, wherein the microorganism is capable of producing fatty acidsin an amount that is at least 30% by weight of the dry cell weight. 20.The isolated microorganism of claim 19, wherein the microorganism iscapable of producing fatty acids in an amount that is at least 40% byweight of the dry cell weight.
 21. The isolated microorganism of claim19, wherein the microorganism is capable of producing fatty acids in anamount that is at least 50% by weight of the dry cell weight.
 22. Anisolated microorganism of the Sporidiobolus pararoseus species, whereinthe microorganism is capable of producing fatty acids in an amount thatis at least 50% by weight of the dry cell weight.
 23. The isolatedmicroorganism of claim 22, wherein the microorganism is capable ofproducing fatty acids in an amount that is at least 55% by weight of thedry cell weight.
 24. The isolated microorganism of claim 22, wherein themicroorganism is capable of producing fatty acids in an amount that isat least 60% by weight of the dry cell weight.
 25. An isolatedmicroorganism of the species selected from the group consisting ofPseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus, andRhodotorula ingeniosa, wherein the microorganism is capable of producinga microbial oil comprising 0.5% to 30% by weight saturated fatty acids,and wherein the microorganism is capable of producing fatty acids in anamount that is 30% to 80% by weight of the dry cell weight.
 26. Anisolated microorganism of the species selected from the group consistingof Pseudozyma aphidis, Pseudozyma rugulosa, Sporidiobolus pararoseus,and Rhodotorula ingeniosa, wherein the microorganism is capable ofproducing a microbial oil comprising 30% to 70% by weight oleic acid and5% to 30% by weight linoleic acid.
 27. An isolated microorganism of theSporidiobolus pararoseus species, wherein the microorganism is capableof producing fatty acids in an amount that is 50% to 80% by weight ofthe dry cell weight, and wherein the microorganism is capable ofproducing a microbial oil comprising 5% to 30% by weight linoleic acid.28. An isolated microorganism deposited under ATCC Accession No.PTA-11615.
 29. An isolated microorganism deposited under ATCC AccessionNo. PTA-11616.
 30. An isolated microorganism deposited under ATCCAccession No. PTA-11617.
 31. An isolated microorganism having thecharacteristics of the microorganism deposited under ATCC Accession No.PTA-11615.
 32. An isolated microorganism having the characteristics ofthe microorganism deposited under ATCC Accession No. PTA-11616.
 33. Anisolated microorganism having the characteristics of the microorganismdeposited under ATCC Accession No. PTA-11617.
 34. An isolated biomasscomprising the isolated microorganism of claim 1 or mixtures thereof.35. The isolated biomass of claim 34, wherein at least 30% by weight ofthe dry cell weight of the biomass are fatty acids.
 36. A culturecomprising the isolated microorganism of claim 1 or mixtures thereof.37. The culture of claim 36, wherein the culture comprises at least 5g/L of biomass of the isolated microorganism.
 38. A method for producinga microbial oil, comprising: growing the isolated microorganism of claim1 or mixtures thereof in a culture to produce a microbial oil.
 39. Themethod of claim 38, wherein the isolated microorganism is grown in thepresence of a carbon source selected from the group consisting ofsucrose, glucose, fructose, xylose, glycerol, mannose, arabinose,lactose, galactose, maltose, cellulose, lignocellulose, and combinationsthereof.
 40. The method of claim 38, further comprising extracting themicrobial oil.
 41. A method for producing a microbial oil, comprisingextracting the microbial oil from the isolated biomass of claim
 34. 42.The method of claim 38, wherein a culture comprising biomass of theisolated microorganism is produced and the culture comprises at least 5g/L of the biomass.
 43. A microbial oil produced by the method of claim38.
 44. Use of the isolated microorganism, biomass, culture, ormicrobial oil of claim 1 for the manufacture of a food, supplement,cosmetic, or pharmaceutical composition for a non-human animal or human.